SILICON NANOSTRUCTURES FOR BIO ANALYTES DETECTION

With GLAD-MACE substrate, we researched the characterization methods, and characterized the substrate surface properties in terms of surface area as well as surface porosity.. Oligonucle

SILICON NANOWIRES FOR THE DETECTION OF

BIO-ANALYTES

CHENG HE

(M.Eng, MIT)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

IN ADVANCED MATERIALS FOR MICRO- AND NANO-

SYSTEMS (AMM&NS)

SINGAPORE-MIT ALLIANCE

NATIONAL UNIVERSITY OF SINGAPORE

2014

DECLARATION

I hereby declare that this thesis is my original work and it has been written by

me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis

This thesis has also not been submitted for any degree in any university previously

CHENG HE

17th Nov 2014

ACKNOWLEDGEMENTS

I’d like to use this opportunity to express my gratitude the people who have helped me tremendously in the past four years

I would like to thank Professor Choi Wee Kiong, who convinced me to continue

my study after my master degree; since then I was introduced into the amazing world of scientific research During the course of my research, Prof Choi has always been most encouraging, helping me to explore unfamiliar subjects despite many trials and errors I am also inspired by his passion and his rigorous scientific reasoning, which have helped me in developing the research work I

am extremely lucky to have Prof Choi as my supervisor, who has not only given

me guidance on my research, but also counselled me to help me to grow

I would also like to thank Professor Too Heng-Phon, who has inspired me to take on challenges that demands hard work I began to appreciate the resilience needed for scientific research, and I am always humbled by his wealth of experience and knowledge We had hours of discussions which made me to think about many things beyond research Prof Too has also brought me into Karate which keeps me fit and refreshed every week during the course of my study

My gratitude also goes to my thesis advisory committee member Prof Eugene Fitzgerald, his encouragement that kept me focused on my goal

The thesis work would not be possible with my colleagues either I would like

to thank Dr Zhou Lihan, who has inspired me with his sharp observations,

to thank Wu Jiaxin, who has worked together with me to study GLAD-MACE for biomarker detection, Zheng Han for collaboration and discussion on GLAD-MACE optimization and the fabrication of the substrates with Xu Wei, Lai Changquan for insightful discussion on MACE and creative ideas for fabrication, Dr Zou Ruiyang for designing oligonucleotide sequences

To my fellow lab mates, Zhou Kang, Zhu Mei, Sarah Ho, Seow Kok Hui, Chin Meiyi, Mai Tong Thi, Khalid, Bihan, Raja, Yudi and Ria, thank you for your companionship

Last but not least, the thesis is especially dedicated to my loving family I am also blessed to meet my wife Chen Xixian in Singapore-MIT Alliance It is great

to meet someone who goes through the same journey and shares the same passion

TABLE OF CONTENTS

DECLARATION i

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iv

SUMMARY x

LIST OF TABLES xiii

LIST OF FIGURES xiv

LIST OF SYMBOLS xix

Chapter 1 Introduction 1

1.1 Motivation and Objective 1

1.2 Organization 2

Chapter 2 Literature Review 5

2.1 Applications of Solid State Nanostructures in Biosensing 5

2.1.1 Solid-state nanopore 5

2.1.2 Fluorescent nanoparticles 6

2.1.3 Nanowires and nanotubes 7

2.1.4 Nanostructured surfaces 9

2.2 Nanoscale Surface Engineering for Microarrays 10

2.3 Probes Immobilization Strategies 18

2.4 Detection and Amplification 25

2.4.1 Amplification technique 27

2.4.1.1 Amplification through bio-affinity systems 27

2.4.1.2 Rolling circle amplification 29

2.4.1.3 Catalysed reporter deposition (CARD) 30

2.5 Sepsis Molecular Biomarker 32

2.6 Conclusion 37

Chapter 3 Fabrication and Characterization of Nanostructured Si Surface ……… 39

3.1 Introduction and Background 39

3.2 Materials and Methods 41

3.2.1 Substrate fabrication 41

3.2.2 Electron microscopy 44

3.2.3 Surface functionalization 44

3.2.4 Dye conjugation 45

3.2.5 Laser scanning 46

3.2.6 Fluorometry 46

3.2.7 Thermoporometry 47

3.3 Results and Discussion 47

3.3.1 Fabrication of GLAD-MACE substrate with Au and Ag 47

3.3.1.1 In-situ estimation of surface area 50

3.3.2 Optimization of Au GLAD-MACE for maximum RFU 54

3.3.2.1 Longer evaporation produced more porous wires 54

3.3.2.2 Optimum etching duration exists 55

3.3.2.3 Oxidation 58

3.3.3 Quantification of surface and porosity 59

3.3.3.1 Determination of absolute area with fluorometry 59

3.3.3.2 Measurement of porosity and absolute surface area with TPM 61 3.4 Conclusion 65

Chapter 4 GLAD-MACE Substrate as Hybridization Platform 67

4.1 Introduction and Background 67

4.2 Materials and Methods 68

4.2.1 Crosslinking of oligonucleotides 68

4.2.2 Hybridization of oligonucleotides 70

4.2.3 Fabrication of fluidic chamber 71

4.2.4 Fluidic setup 73

4.3 Results and Discussion 74

4.3.1 Limit of detection 75

4.3.1.1 Detection of miRNA 78

4.3.2 Factors influencing hybridization 80

4.3.2.3 Quantification of equilibrium 84

4.3.2.4 Limitation by bulk diffusion 88

4.4 Conclusion 96

Chapter 5 Photo-attachment of Biomolecule for Miniaturization on GLAD-MACE Substrate 98

5.1 Introduction and Background 98

5.2 Materials and Methods 100

5.2.1 Photoactivable moiety attachment 101

5.2.2 Liquid phase chromatography mass spectrometry 102

5.2.3 Surface passivation 103

5.2.4 Photo-attachment 103

5.3 Results and Discussion 104

5.3.1 Wicking behaviours of GLAD-MACE substrate 104

5.3.2 Approaches of photo-attachment 106

5.3.3 Miniaturization through photo-attachment 110

5.3.4 Comparison of photo-attachment to chemical crosslinking 113

5.3.5 Effect of photo-attachment buffer and surface area 115

5.3.6 Influence of static charge interaction 117

5.3.7 Hybridization 119

5.3.8 Influence of probe structure 122

5.4 Conclusion 127

Chapter 6 DNA Directed Addressing for Cytokine Detection 128

6.1 Introduction and Background 128

6.2 Materials and Methods 129

6.2.1 Protein labelling with fluorophore 129

6.2.2 Buffer exchange 130

6.2.3 Measurement of protein and oligonucleotide concentration 130

6.2.4 Synthesis and optimization of protein-oligonucleotides conjugates 132

6.2.5 Mobility retardation assay 135

6.2.6 Enzyme-linked immunosorbent assay 137

6.2.7 Immunoassay on chip with DDA 138

6.2.8 Tyramide signal amplification 138

6.3 Results and Discussion 139

6.3.1 Oligonucleotide conjugation maintains activity of antibody 142

6.3.2 Capturing of model molecule with DDA 144

6.3.3 Sandwich assay with DDA 146

6.3.4 Reduction of background 150

6.3.5 Optimization for hybridization 154

6.3.6 Detection of selected biomarkers in human serum 158

6.4 Conclusion 161

7.1 Future Work 165

7.1.1 Active driving for addressing 165

7.1.2 Robust multiplexing 167

7.1.3 Label free detection 168

REFERRENCES 170

Appendix A List of Publications 206

SUMMARY

This thesis studies the application of Si based nanowires produced from metal assisted chemical etching (MACE) for the detection of biomolecules We surveyed various nanotechnologies and their merits in the biomedical and life science research, and investigated the technical aspects of incorporating nanostructures with microarray and biosensor technology

We first showed the fabrication of high aspect ratio dense nanowire substrate through gold catalysed etching on silicon substrate Glancing Angle Deposition (GLAD) was used to enable the formation of gold catalyse nano-clusters The production process was simple and of low cost compared to other nanowire forming procedures With GLAD-MACE substrate, we researched the characterization methods, and characterized the substrate surface properties in terms of surface area as well as surface porosity We found the combination of both factors increased the available surface sites for subsequent chemical surface modification, which is necessary for transforming the nanowires into a biofunctional solid substrate In addition, various optimization was performed with the aim to achieve better surface loading capacity

Subsequently, we investigated the possibility of using GLAD-MACE nanowires as DNA hybridization platform Oligonucleotide DNA was chemically grafted to the nanowire surface and we are able to achieve a surface loading density of oligonucleotides two orders of magnitudes higher than that

of conventional surface without inflicting on hybridization efficiency often observed from the increased steric hindrance from conventional solid substrate

miRNA, and demonstrated that the limit of detection can be extended at least

10 folds lower compared to the results obtained on conventional substrate based

on similar detection methodology Furthermore, we showed the substrate has an excellent discrimination power against oligonucleotides with single nucleotide mismatch In addition, we investigated other factors that may influent the detection of oligonucleotide, including overall surface hybridization equilibrium as well as mass transport We concluded that it would be of a great importance to allow faster mass transport for better detection of oligonucleotides

We have also researched the miniaturization of oligonucleotide functionalization on GLAD-MACE nanowires Functionalized with hydrophilic coatings, such nanowire substrate becomes superhydrophilic, where the printing

of smaller sensor detection sites becomes increasingly difficult Yet, it is often desirable to decrease the dimension of detection sites so that the signal intensity

as well as throughput may increase We showed that photo responsive surface can be patterned through the use of photoactive reagent, such patterning enables

us to achieve smaller detection spot which cannot be achieved even with the best commercially available piezo printers The mismatch discrimination and detection are not compromised with photo-activation The technique represent

a significant cost saving which can facilitate the adaption of superhydrophilic materials for miniaturization purposes

Lastly, we modified DNA directed immobilization (DDI) method to DNA Direct Addressing (DDA) for the production of a protein detection platform on GLAD-MACE nanowire substrate Protein is a class of important functional molecules that can provide deep insight in biomedical research We showed the

proof-of-concept platform that can be used for cytokine detections in human serum which is potentially useful in disease (e.g Sepsis) diagnosis We have carried out numerous optimization strategies so that the limit of detection can

be pico Molar range

In conclusion, we showed that GLAD-MACE nanowires showed excellent characteristics with the various applications demonstrated here The thesis laid the ground work for their future development

LIST OF TABLES

Table 3.1 Comparison of amount of conjugated fluorophores, GLAD-MACE conjugated significantly more fluorophores per footprint compared to flat

wafer 61

Table 6.1 Various parameters used for absorbance measurement 132

Table 6.2 Oligonucleotide sequences 134

Table 6.3 Antibody and antigens information 135

LIST OF FIGURES

Figure 2.1 A nanowire FET device for biosensing Reprinted from Ref5, with permission from Elsevier 8Figure 2.2 Dendrimers forming nanobumps on glass surface from Ref63, Reprinted with permission Copyright (2014) American Chemical Society 12Figure 2.3 Various nanopillars fabricated on quartz, reprinted from Ref68, Copyright 2014, with permission from Elsevier 14Figure 2.4 The use of nanostructured substrate to facilitate microarray

fabrication, reprinted with permission from Ref76 (A) Above: Comet tail defect from printing and Below: Nanostructures prevents the forming of comet tail defects (B) Left and Middle: Donut defect on hydrophilic substrate, Right:

no defect on hydrophobic substrate 17Figure 2.5 Parallel in-situ synthesis of oligonucleotide probes, reprinted from Ref112 with permission from Nature Publishing Group (A) Light deprotect the surface group to expose hydroxyl group for nucleotide addition (B) mask set that is used for location specific addition of nucleotide 24Figure 2.6 Set up of an SPR detection system, reprinted from Ref119, with permission from WILEY-VCH 26Figure 2.7 Schematics of Layer by layer assembly of biotinylated protein networks for signal amplification Reproduced from Ref131 with permission of The Royal Society of Chemistry 28Figure 2.8 TSA scheme (A) signal generation without amplification, (B) HRP catalysed conversion of TSA-fluorophore conjugates and (C) signal

amplification through immediate crosslinking of tyramide radical with protein 32Figure 2.9 Receiver operating characteristics (ROC) of PCT for adult

Reprinted from Ref180, with permission from Elsevier 35Figure 2.10 Various molecules investigated as biomarkers for sepsis, reprinted from Ref182 37Figure 3.1 Interference lithography setup (A) Placement of key components, substrate was rotated 90 degrees after the first exposure, and (B) Illustration of interference pattern on substrate after 2 exposures 42Figure 3.2 Producing ordered nanowire array using IL and MACE, Ref219 43Figure 3.3 Glancing Angle Deposition of gold nanoclusters (A) gold influx

Figure 3.4 Nanoclusters produced by GLAD (A) Au deposition, (B) Ag deposition, (C) etched nanowires from Au deposition, (D) etched nanowires from Ag deposition (E) TEM image of the middle section of Au etched

nanowire and (F) TEM image of the middle section of Ag etched nanowire 49Figure 3.5 RFU values of different substrate, gold GLAD-MACE substrate produced highest RFU 52Figure 3.6 Gold quenching of fluorescence reading 1 Hr gold removal time showed significant higher RFU compared to gold removal for 1 minute and 2 minutes 54Figure 3.7 Effect of etching duration (A) Nanowire clumps from 4 mins etching, (B) Nanowire clumps from 10 mins etching, (C) nanowire length verses etching time and (D) RFU values for nanowires of different lengths 57Figure 3.8 Oxidation and surface RFU 35 minutes of oxidation time resulted

in highest RFU after fluorophore conjugation 58Figure 3.9 (A) Calibration of emission of Cy3 in the presence of detergent and (B) standard curve of fluorophore emission 60Figure 3.10 Schematic drawing of pore ice melting, additional interface elevates the total energy of ice inside pores thus depressing its melting point 62Figure 3.11 Pore size measurement of Au GLAD-MACE nanowires 63Figure 3.12 Scratched off nanowires, insert shows a much smaller bunch compared to unscratched nanowires which forms clumping 64Figure 4.1 (A) Fabrication procedure for microfluidic chamber and (B) top view of the pattern of microfluidic chip 73Figure 4.2 Fluidic equipment set up Syringe pump was used to generate a pressure to push hybridization liquid through PTFE tubing to flow under a housing bound on chip 74Figure 4.3 LoD of GLAD-MACE nanowires for oligonucleotides target Insert: Low concentration conditions 75Figure 4.4 Detection of hsa-let-7a and single mismatch discrimination on GLAD-MACE substrate 79Figure 4.5 Crosslinking of oligonucleotides on GLAD-MACE nanowires (A) Comparison of crosslinking on GLAD-MACE and flat substrate, (B) RFU reading of GLAD compared to flat, (C) crosslinking of oligonucleotides with beads of different pore sizes, (D) absolute amount of crosslinked probes and (E) the RFU readings of crosslinked probes on substrate 81Figure 4.6 (A) Scanned image of hybridization signal on sense

oligonucleotides functionalized substrates, and (B) RFU readings of

hybridization 84

Figure 4.7 Remaining target concentration in reaction volume over time 87

Figure 4.8 Flow incubation compared with free incubation (A) Incubation with 0.1 μM target and (B) incubation with 1 μM target and (C) 0.1 μM target antisense incubation with slower flow rate 89Figure 4.9 The effect of mean velocity on boundary layer and target capturing (A) model of the bulk diffusion and (B)the predicted results 91Figure 4.10 13.3 μL/ml flow in 50 um chamber compared to stationary

incubation 93Figure 4.11 Experiment RFU for fast and slow flow rate for 50 μm chamber, faster flow produced much higher reading (blue dots) compared to slower flow (red dots) 95Figure 5.1 Top view of nanowire substrate (A), side view of nanowire

substrate (B), scale bars are of 20 μm, (C) the spreading of liquid doped with fluorophores on GLAD-MACE substrate (D) the wicking model and (E) the wicking behaviour measured with regard to time, the trend line shows data fit for hemiwicking diameter with respect to t0.25 105Figure 5.2 LCMS of modified and unmodified oligonucleotiedes (A) Intensity

vs Time graph for oligonucleotide control, (B) Intensity vs Time graph for modified oligonucleotides (C) peaks for modified oligonucleotides and (D) unmodified oligonucleotides 108Figure 5.3(A) Chemical reaction approach for photo-attachment of

biomolecules on nanowire surface and (B) simple set up for photo-attachment The black parts in Fig 1B represents opaque region of photomask The yellow region represents photo-attached nanowires 110Figure 5.4(A) Array Fabrication of photo-attached spots of various diameter, yellow arrow pointing anomaly surface area resulted from nanowire

fabrication process (B) Schematic diagram showing the approach to

immobilize different probes at different positions, 4 different probes can be immobilized with replicates at one go, and (C) piezo printing by Scienion AG with 100 and 50 pico Liter dispensing volume, with permission from Scienion

AG 112Figure 5.5 Comparison of crosslinking efficiency between chemical

immobilization and photo-attachment 114Figure 5.6 Photo-attachment to crosslink sense oligo on nanowire substrates (A) Psoralen functionalized nanowire substrate, (B) Diazirine functionalized nanowire substrate, (C) Psoralen functionalized polished silicon substrate with the inset shows the same chip s scanned at maximum power and PMT gain, (E) Psoralen functionalized with passivated substrate, and (F) Diazirine

Figure 5.8 Hybridization of target oligo on photo-attached substrates (A) Psoralen functionalized substrates, (B) Diazirine functionalized substrates, (C) Psoralen functionalized substrate with passivation, and (D) Diazirine

functionalized substrate with passivation 120Figure 5.9 (A) Limit of detection, (B) Single mismatch discrimination without formamide and (C) Single mismatch discrimination with formamide 122Figure 5.10 Probe crosslinking on nanowires with different probe structure (A)

on psoralen functionalized surface passivated with adipic acid and (B) on diazirine functionalized surface passivated with adipic acid 123Figure 5.11 Photo-attachment on (A) Psoralen/COOH surface and (B)

Diazirine/COOH surface at wavelengths of 250 nm, 302 nm and 365nm with a probe concentration of 20 µM (C) and (D): Hybridization on nanowires crosslinked with 20 µM probes, (E) and (F): Hybridization on nanowires crosslinked with 2 µM probes, Note that the bars indicate sense crosslinking and the lines indicate hybridization 126Figure 6.1 Mobility retardation assay for oligonucleotides and protein

conjugation, insert shows non-optimized condition where the yellow arrow points to unconjugated protein 136Figure 6.2 Schematic illustration of DDA with GLAD-MACE substrate 1 Amination with APTES, 2 Carboxylation with adipic acid 3 Sense

functionalization 4 Homogenous phase capturing by ASR and 5 DDA of ASR on nanowire 141Figure 6.3 The effect of oligonucleotide conjugation (A) Measurement of limit

of detection before conjugating on Anti human IL-8, (B) measurement after conjugating Anti human IL-8, (C) same conjugation on enzyme Erg12 and (D) IgG modification sites 144Figure 6.4 DDA on sense functionalized GLAD-MACE substrate (A) Signal generated from a flat silicon chip and (B) the RFU values compared 146Figure 6.5 Sequential Assembly versus One step assembly Sequential

assembly showed a much higher SNR (9.8) compared to that of One step assembly (2.74) 148Figure 6.6 Validation of sandwich assay on DDA substrate with IL-8 (A) IL-8 detection above 8 ng/ml (1nM) showed good signal response (B) and (C) shows IL-8 detection below 1 nM, where no trend could be observed in the detection response 149Figure 6.7 Optimization for reducing nonspecific adhesion.(note the PMT gain and power settings are different for A,B and C) (A) Effect of BSA blocking

on sense functionalized sites and carboxylated surface, (B) Effect of BSA blocking on sense functionalized sites for ASR hybridization, (C)

Optimization of other factors and (D) Detection of IL-8 after the reduction of background 153

Figure 6.8 Effect of salt concentration on Hybridization (A) ASR

hybridization time response, (B) RFU response surface with monovalent and divalent anions and (C), improved detection limit with high salt concentration 155Figure 6.9 Matrix effect inhibits the detection of analyte in human serum 159Figure 6.10 TSA amplified detection of cytokines in serum 161Figure 7.1 Active analyte driving with electrical field, (A) schematic drawing and (B) driving results 166Figure 7.2 An EIS sensor embodiment with the GLAD-MACE substrate, with

in plane detection scheme suggested by Ref441 169

ASR Analyte specific reagent

CARD Catalysed reporter deposition

EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride EDTA Ethylenediaminetetraacetic acid

GLAD Glancing angle deposition

HPLC High performance liquid chromatography

IL-6,8,10… Interleukin-6, 8, 10…

LC MS Liquid chromatography mass spectrometry

MACE Metal assisted chemical etching

miRNA microRNA

RCA Rolling cycle amplification

RFU Relative fluoroscent unit

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate SNR Signal to noise ratio

SPB Succinimidyl-[4-(psoralen-8-yloxy)]butyrate

CHAPTER 1 INTRODUCTION

1.1 Motivation and Objective

Nanostructured materials defer from their bulk counter parts in many physical and chemical properties Shrinking of the dimension changes fundamentally the interactions of materials with their surroundings Various mechanisms, such as the confinement of charge carriers,1 the increase in surface to volume ratio2 and the comparable material length scale to visible spectrum3,4 contribute to their distinct characteristics The unique qualities of nanostructured materials can be exploited for construction of devices with performance that cannot be matched with bulk materials.5,6 As a result, nanostructured materials have been found in many exciting innovations not only in material sciences, but also in scope beyond material research

The landscape in biomedical research has been greatly altered by the fusion of nanotechnology with biotechnology In biomedical studies, researchers are often concerned in the detection of analyte of interest contained in samples to extract information about the state of a biological system.7 Such information is useful not only for the scientific understanding of living organisms, but also can

be applied in healthcare industries for disease prevention and diagnosis, and as guidance for treatment However, the accurate detection of these analytes is often challenged by their scarcity in biological system,8 where signal of detection can often be flooded by noise and no meaningful interpretation can be derived.9 To solve the problem, many nanostructured materials, such as

quantum dots,10 nanowires11 and nano porous materials12 were introduced to increase the detection sensitivity

Mesoporous silicon possess pores in the range of 2 to 50 nm and can be fabricated with glancing angle deposition (GLAD) of metal catalysts and metal assisted chemical etching (MACE) method The nanostructures can be produced without the use of lithography and sophisticated etching techniques such as reactive ion etching (RIE), thus making GLAD-MACE a cost effective approach to produce nanostructures on silicon surface Therefore, it is interesting to investigate the application of GLAD-MACE structure in biomedical research; as the relatively simple fabrication process would offer an inexpensive option for higher performance required for sensitive detection of bio-analytes We aimed to gain a thorough understanding of the material itself, the understanding would provide solid foundation for the application We looked into two different categories of most commonly encountered biomolecules, namely, nucleotides and protein, to show proof-of-concept device can be made on GLAD-MACE nanowires as a versatile platform in general

1.2 Organization

The thesis will be organized into 6 subsequent chapters

Chapter 2 covers briefly the nanostructures that have been widely used in biomedical research field The discussion aims to paint a broad picture of

MACE technique We will also review other technologies that are essential to produce a biosensor or microarray, namely, the technology for probe immobilization, for bioconjugation, for detection and amplification Lastly, some potential biomarkers that can be used for sepsis diagnosis was reviewed

Chapter 3 covers a more physical side of the topic, where the fabrication and optimization of the GLAD-MACE nanowires will be discussed Furthermore, the characterization of the nanowire surface will be discussed One essential parameter, unit surface area, will be quantified Another parameter, the porosity

of the nanowires, will also be investigated through the use of thermoporometry

Chapter 4 discusses the basics of oligonucleotide probe immobilization The surface loading capacity for these larger molecules will be investigated and compared to conventional substrates Furthermore, the detection limit of target oligonucleotide, the single nucleotide mismatch discrimination were also investigated

Chapter 5 concerns more about the practical aspect of producing a miniaturized device on GLAD-MACE nanowire substrates Nanostructured surface, when functionalized with hydrophilic functional layers, tends to become superhydrophilic As a result, controlled immobilization is difficult to achieve

We demonstrated a strategy to immobilize oligonucleotides with photon directed synthesis We achieved smaller dimensions with photo attachment compared to state-of-the-art piezo nozzle technique and achieved significant cost reduction for fabrication

Chapter 6 discusses the method of transforming DNA detection platform to a protein detection platform through the technology of DNA-directed addressing

(DDA) Model molecule will be used to show the conceptual realization of DDA

on GLAD-MACE, while the capture and optimization of detection of cytokines that can be potentially used as sepsis biomarkers will be shown

Finally, Chapter 7 summarizes the accomplishments in this study and provide recommendations for future work

CHAPTER 2 LITERATURE REVIEW

Nanotechnology is the science and engineering to design, fabricate, characterize materials and structures with at least one dimension smaller than 100 nm.13During the past decade, nanotechnology has made great strides in various fields

of applications, especially in the field of life sciences to bring the advances in engineering to the study of biology In this chapter, a wide range of applications

of nanostructures in biological fields will be surveyed first Subsequently, nanotechnology used to engineer microarray substrate will be reviewed; with special focus the technological aspects of microarray, e.g probe immobilization, signal readout and amplification Finally the biomarkers used in this work will

be reviewed with a detailed discussion on the choice of these biomarkers for real life application

2.1 Applications of Solid State Nanostructures in Biosensing

Recent advances in nanotechnology has enabled nanostructures to be used in biological applications, such integration makes it possible to build advanced tools to reveal and study the properties of a biological system on molecular basis Nanopore, nanoparticles, nanowires as well as nanostructured surfaces are among the most frequently used nanostructures based on solid state materials

2.1.1 Solid-state nanopore

Biological nanopore has been extensively researched for the use of sequencing, DNA profiling as well as molecular diagnostics.14 Although biological nanopores have been shown to possess remarkable sensitivity, they have inherit

weakness in terms of mechanical and chemical stability On the other hand, solid state nanopores are gaining research interest because they offer advantages

in terms of robustness, precision, throughput and ease of integration.15 The first solid state nanopore was built with Ar ion beam sputtering on Si3N4

membrane,16 The authors observed a current blockage occurred when DNA translocate through the nanopore Since then, other materials have been experimented for building solid state nanopores to improve the resolution of the pore device For example, single or double layer graphenes were used to fabricate nanopore device because of its remarkable homogeneity in thickness, which is comparable to the dimension of single nucleotide.17 Nanopore device based on graphene is able to differentiate double strand DNA (dsDNA) with different folded structures It is estimated by reducing DNA translocation speed, single nucleotide differentiation is possible.14 Solid state nanopores have also been employed in microRNA (miRNA) measurement,18 single nucleotide polymorphism (SNP) detection19 and genomic profiling.20

2.1.2 Fluorescent nanoparticles

Nanoparticles belong to another category of zero dimension nanostructure that has seen a wide array of applications Two major applications in nanoparticles include imaging and drug delivery For imaging, semiconductor particles in nanometre scales are used due to their strong quantum confinement effect; such particles are termed as quantum dots These quantum dots are typically fabricated with II-VI or III-V semiconductor materials,21 where the precursor is injected into heated organic solvent to form particle cores Subsequently, a large

dots have been increasingly used to replace organic fluorophores The most direct advantage of quantum dots is their much brighter fluorescence (around

20 fold22,23) due to higher extinction coefficient and comparable quantum yield compared to conventional molecular fluorophores.24 The excitation and relaxation of quantum dots can also be tuned by changing their size and chemical composition, making them suitable for multiplexed assay Furthermore, quantum dots are much more photostable compared to molecular fluorophores, the stability help to extend their use in situations where prolonged observation times is needed In addition to imaging applications, nanoparticles can also be used for molecular beacons,25 drug carriers,26,27 or even be therapeutic agents themselves.28,29

2.1.3 Nanowires and nanotubes

One dimensional nanostructures such as nanowires and nanotubes can be used for labelling agent and sensors Segmented metallic nanowires can be used for barcoding,30 by arranging of segments in different orders a large number of different analytes can be encoded.31 Such labelling scheme can overcome the limitations of fluorescence labelling, where the inadequate number of fluorophores with different fluorescence has placed severe limitations on the multiplex capability of bioassays, not to mention these nanowires or nanotube based labelling are more stable than fluorophores.32 A more explored area for one dimensional nanostructure, however, is label free sensors which harness the unique properties nanowires to increase the sensitivity of detection Although sensors that use nanowires/nanotubes as electrodes exist,33,34 most nanowire/nanotube based sensors are configured into field effect transistors (FETs) based on semiconducting materials, as such materials offers great

freedom in tuning their electrical properties.35 Figure 2.1 shows a schematic diagram of nanowire FET sensor The sensitivity of FET based sensors can be further configured by changing the biases between gate, source and drain.36When a biomolecule is attached to the nanowire/nanotube sensor, a small change of surface charge distribution occurs Due to exceptionally diminutive diameter of nanowires, the variation in surface charge causes a large change in the depth of depletion/reversion layer between source and drain,37 resulting in a shift of gate threshold voltage and an exponential change in saturation current

As such, it is desirable that the nanowire diameter is comparable to the Debye layer thickness of the biomolecule inside the nanowire, so the effect of accumulation/charge reversion layer is more pronounced.35 In addition, nanowire FET sensors also require the conductivity of the wires to be much larger than that of the buffer solution, so that less current leaks from nanowires.38 As such, the design of the nanowire FET sensor ideally requires low carrier concentration, high carrier mobility as well as small diameter

Figure 2.1 A nanowire FET device for biosensing Reprinted from Ref5, with

Although one dimensional nanowire sensors are very sensitive, they still face some limitations Under physiological conditions, metal oxide nanowire is often unstable,35 and Si nanowire forms an oxide layer which decreases nanowire sensitivity Furthermore, surface modifications essential for attaching biomolecules often result in an increase in the distance between biomolecules and surface, or in the case of carbon nanotube, the production of defect which impair charge carrier mobility.39 It is also noted that the nanowire sensors need

to operate under low salt conditions,40 so that the shielding effect of buffer does not decrease the sensitivity, however, such low salt conditions are not optimum for biomolecules interactions.41

2.1.4 Nanostructured surfaces

Nanostructured surfaces play important role in biomedical research because such surfaces take advantages of the unique properties of nanomaterials, while still being suitable for large-area applications Nanostructured surface can be fabricated on polymer,42,43 oxide,44 metal45,46 as well as semiconductors.47 In biomedical area, such surfaces are mainly used for their properties of restricting

of mass transportation, increased surface area, or for the mechanical stimuli they impose on cells For example, surfaces grown with nanowires and nanopillars have been used for separation of biomolecules between DNA and miRNA.48The separation is achieved through the molecular size sieving effect which restrict the mass transport of large biomolecules.49 The increased surface area

of nanostructured surfaces can also be used for cell capturing, where the enhanced surface interactions help to retain cells of interest.50,51 Furthermore, substrate with increased surface area have been widely used as solid supports for enzymatic reactions as well as detection platform For example, researchers

have shown a 44 fold increase in enzymatic activity due to surface capacity increase when glucose oxidase was immobilized onto a hierarchical micro-nanostructured surface.52 Based on the same principle, we have shown recently

a nanostructured surface formed by metal assisted chemical etching can enhance the surface loading capacity by 100 fold, thus improving detection limit for microarray devices.53 In addition, nanostructured surfaces have been used to guide cell development and differentiation, for the nanostructures can interact with cell focal adhesion (FA) mechanism which transduces mechanical signals into biochemical events.54 For example, varied stem cell differentiation has been observed on nanostructured surfaces with different patterns,55 leading researchers to build a microarray with a collection of nanopatterns to help stem cell differentiation into a wide lineage for drug screening purposes.56,57 We have recently demonstrated nanogrooves patterned surfaces can induce neurite guidance through miRNA interactions.58

In summary, the progress in nanotechnology has bestowed researchers with unprecedented capability of producing materials and devices in nanometer scale, such devices and materials have greatly impacted biomedical and life science research in many fronts The subsequent sections will focus on nanostructured surface may improve current technology of microarray or biosensor

2.2 Nanoscale Surface Engineering for Microarrays

Microarrays are lab-on-a-chip solid phase detection devices with multiplex capability Based on their target analytes, microarray can be employed for the

Among these, nucleotides microarrays (cDNA microarray and oligonucleotide microarray) are relatively mature where they have been used for tasks such as genetic expression profiling, single nucleotide polymorphism (SNP) detection and disease diagnosis Protein microarrays, which probes proteomics information, facilitate protein functional study and disease diagnosis, were inspired by nucleotide microarray and have seen a recent surge in development

For any microarray, solid substrate is always one of the most vital component, not only because it affects immobilized biomolecules in terms of activity and stability, it also affects multiple aspects in microarray printing and signal readout59 Much engineering effort has been invested to produce substrate with for enhanced signal readout and probe immobilization

It is observed that fluorescence readout can be affected by surface oxide nanostructures Since many microarrays are produced on oxide ranging from SiO2 to Al2O3,60 optimization of oxide layer on top surface of the solid support

is one significant task Bras et al have found oxide layer thickness affects

optical signal intensity on a planar solid support.61 It is observed that the fluorescence readout fluctuates with oxide thickness ranging from 1 to 500 nm For commonly used fluorophores such as Cy3 and Cy5, signal intensity peaks around oxide layer thickness of 100 nm The authors concludes that such fluctuation is due to the interference between oxide layer and the underlying Si layer Such effect is not just limited to planar surface On surface with nanostructures, oxide layer on top of the nanostructures can still induce the interference effect Different from planar surface though, the underlying nanostructures change the interference peaks For example, on nanobumps coated with SiO , the emission peak of Cy3 is found to be 50 nm rather than

100 nm,62 indicating importance of optimization of oxide for individual type of solid support

As mentioned in Section 2.1.4, nanoscale engineered surface can also be used

to increase the surface capacity of the microarray Various schemes have been proposed Researchers have tried to assemble poly(propyleneimine) dendrimer terminated with carboxyl group (PAMAM-COOH) on top of glass slides.63 As shown in Figure 2.2, such dendrimers forms nanoscaled bumps on glass slide, resulting in a roughened surface to provide increased surface area Furthermore, such dendrimer showed very low nonspecific adhesion towards bioanalyte, which is useful for reducing background

Figure 2.2 Dendrimers forming nanobumps on glass surface from Ref63, Reprinted with permission Copyright (2014) American Chemical Society

Based on the same principle, nanotextured surfaces have been fabricated on polymer like polystyrene (PS)64and silicon to increase the immobilization density of DNA or protein.65,66

It is worth noticing, the nanostructures on the surface do not only promote probe

example, Bong et al have found that solid substrate with meso-scaled

nanostructures promotes SNP discrimination,67 with the discrimination ratio similar to that in the solution It is suggested by the authors that such nanotextured surface can separate oligonucleotides from adjacent strands as well as the bulk solid surface, allowing the molecules higher degree of freedom,

as if the molecules are dissolved in the solution

Although nanotextured surfaces have shown advantages to increase surface area, they are far from being ideal because these textures are often of low aspect ratio, thus the surface area increase is relatively small To increase the surface area, high aspect ratio structures are needed Figure 2.3 shows various nanopillars fabricated on quartz with aspect ratio around 5 through maskless patterning and etching with RIE.68 The nanopillar substrate was used for fluorescence detection

of oligonucleotides, and it showed a 6 fold improvement in SNR and a slight

improvement in mismatch discrimination Similarly, Murthy et al produced a

microarray substrate with ordered nanopillars with aspect ratio greater than 10 through reactive ion etching (RIE).69 They found that Si nanopillars with an oxide layer on top produced 7 fold higher signal to noise ratio (SNR) compared

to flat substrates The SNP discrimination on the nanopillar substrates was also around 3 fold better Similar technologies have also been used for protein detection with improved sensitivity.70

Figure 2.3 Various nanopillars fabricated on quartz, reprinted from Ref68, Copyright 2014, with permission from Elsevier

Although nanopillar arrays have shown improved sensitivity compared to planar substrate, their fabrication requires etching in the vacuum, which substantially increased cost and production time In addition, the patterning process to fabricate nanopillars often involve the use of lithography technique (e.g nanosphere lithography71) which often limits the feature density High aspect ratio nanowires solid support which can be fabricated through bottom up

approaches have been researched to solve the issues Serre et al demonstrated

the fabrication of Si nanowires through vapour liquid solid (VLS) growth to produce microarrays.72 Although the substrate production still required vacuum environment, the density of the nanowires can be much higher than those of nanopillars In addition, VLS nanowires are of very high aspect ratio, thus can greatly increase the available surface area for biomolecule attachment As a

simplify the fabrication process, we have recently devised a fabrication method where closely packed nanowires can be fabricated through glancing angle deposition of gold and metal assisted chemical etching.53 This substrate showed significant increase in surface area both due to the 3D protrusions of nanowires,

as well as the porous sidewalls of nanowires As a result, a 250 fold increase in signal intensity of immobilization and hybridization can be observed

In addition to the enhanced signal intensity of nanostructured solid support, substrates with nanoporous structures can also be employed for better control

of immobilization homogeneity of biomolecules As shown in Figure 2.4A, the nanostructures can also prevent excessive molecules from leaving surface in the washing step at the end of immobilization

For microarrays, it is desirable to increase the immobilized biomolecules on the surface However, a simple increase in microarray printing volume will result

in large spreading of printing buffers on the array surface Thus, superhydrophobic substrate can be used to contain the liquid while increasing microarray printing volume, thus a large number of probes can be immobilized into a small spot Such technique resulted in protein microarrays with high sensitivity down pico molar range.73

Furthermore, superhydrophobic nanoporous substrate can also facilitate the control of microarray size and spot homogeneity as shown in Figure 2.4B First, the well-known “coffee stain effect” is often encountered in microarray fabrication.74 The effect is known to increase the variation between spots as well

as reducing the reproducibility of microarray experiments.75 Although the effect can be mitigated by heating and addition of organic solvent for printing more

stable biomolecules, it remains challenge for producing microarrays that contain more fragile molecules Figure 2.4B shows the pinning of droplet on a superhydrophobic substrate, which reduces evaporative convection, eliminating the coffee ring surrounding a detection spot

Figure 2.4 The use of nanostructured substrate to facilitate microarray fabrication, reprinted with permission from Ref76 (A) Above: Comet tail defect from printing and Below: Nanostructures prevents the forming of comet tail defects (B) Left and Middle: Donut defect on hydrophilic substrate, Right: no defect on hydrophobic substrate

As the application of microarray continues to broaden, it is expected that the application of nanostructures to microarray for the enhanced immobilization, sensitivity and reproducibility However, there are other important aspects of microarray technology which heavily relies on biochemistry Only by combining the knowledge of these fields can one hope to achieve breakthrough for better devices These aspects will now be reviewed in the next section

2.3 Probes Immobilization Strategies

Various substance, including living cells, inorganic chemicals and cell lysate can be immobilized on biosensing platform, however, protein and nucleic acids immobilization are most commonly encountered

2.3.1.1 Protein immobilization techniques

Interfacing proteins to solid substrate is one of the central themes in producing functional bio-devices However, protein interfacing is a much more complicated problem compared to the likes such as nucleotide The challenges mainly stem from the heterogeneous properties of proteins, where protein molecules vary greatly in amino acid composition, size and hydropathy.77,78 Moreover, unlike nucleotides, proteins easily lose their bioactivity through denaturation due to surface interaction, dehydration and oxidation.77 As such, a variety of techniques are developed to place protein on a solid support while maintaining their functionality Conventionally, proteins could be immobilized

to solid surface through a number of ways The simplest way of attachment is through physisorption where protein is attached non-covalently by hydrophobic

physisorption faces severe limitations, namely, the adsorption is highly dependent on surface properties such as roughness, porosity and electrostatic charge,82 so that physisorption is a highly complex process which needs fine tuning for individual surface and protein type The resultant immobilized proteins tend to be randomly oriented to minimize the repulsive force;81 but it leads to decreased efficacy for other molecules to interact with immobilized proteins Another drawback of physisorption is biomolecules tend to desorb during washing procedures As such, immobilization of proteins tend to be achieved by covalent means which does not only provide much stronger bonding between protein and surface, but also present an approach with better control of immobilization

Covalent immobilization can be achieved by linking of accessible side functional groups of naturally occurring amino acids to substrate surface.83 For example, the residual of lysine provides free amine groups for reaction,84 the residual of cysteine provides thiol groups,85 serine residual offers hydroxyl group and aspartic acid as well as glutamine present carboxyl groups that can

be coupled.81 Substrate surface can be modified with reactive functional groups

to react with above mentioned side chain groups Covalent immobilization through side chain functional groups are often random, since it is difficult to select a specific side chain functional group for immobilization if multiples of the same functional groups are presenting on the exterior of protein As a result, covalent immobilization through side chain often results in protein attachment through multiple residues, restricting the conformational freedom and decreasing activity of immobilized protein.83 A variant of chemical crosslinking utilizes site specific immobilization through specially conjugated functional